Supremerun

pGL3-Promoter, pGL4.10[luc2], pGL4.24[luc2P/minP], and pNL1.1TK[Nluc/TK] vectors were purchased from Promega (Madison, WI, USA). 5′ flanking sequence of the TRIP6 was taken from Ensembl genome browser (Homo sapiens GRCh38.p12) for TRIP6-201 transcript (ENST00000200457.9). Sequence −936/+111 (where +1 means TRIP6 transcription start) was amplified from MCF-7/PacR genomic DNA by PCR and cloned into pGL3-Promoter vector via KpnI and NcoI sites. The inserted sequence was subcloned by PCR into pGL4.10[luc2] and pGL4.24 vectors (Figure S5). Mutagenesis of the CRE motif was achieved by cleavage with AatII-HF enzyme followed by 3′ overhangs removal (Large Klenow Fragment, NEB). The construction of plasmids, primers and PCR conditions are summarized in Figure S5 and Tables S4–S8. The constructs have been verified by restriction endonuclease cleavage and insert sequencing (LightRun, SupremeRun, Eurofins Genomics, Ebersberg, Germany).

All animals were genotyped within the first 2 weeks of life. Genomic DNA was prepared from cheek swabs (GeneJET genomic DNA kit, Thermofisher) and used in polymerase chain reaction (PCR) with primers spanning the splice donor site of dystrophin exon 50 (see
Table 1). PCR products were purified (QIAquick, Qiagen) and submitted for Sanger sequencing (SupremeRun, Eurofins) using the forward primer only. Animals were then either rehomed (WT males and females, spayed carrier females), kept for colony maintenance (carrier females) or enrolled onto the natural history study (WT and DE50-MD: male dogs only) accordingly (see
Figure 1A).